1. Field of Invention
The present invention relates to the field of biopharmacy, and in particular to a modified recombinant human endostatin and application thereof.
2. Related Art
With the viewpoint of “tumor growth dependence on neoangiogenesis” proposed by Professor Folkman in 1971, it is possible to control tumor growth by blocking neoangiogenesis, to achieve the object of inhibiting tumor invasion, recurrence and metastasis, thus creating a new field in tumor therapy.
In 1997, it was found by O'Reilly, et al. in Harvard University that, the culture medium of mouse hemangioendothelioma endothelial cells had inhibitory effect on endotheliocytes, and by purification, such active species exhibited dose-dependent growth inhibition endotheliocytes specifically in a certain concentration range (100 ng/ml-600 ng/ml). It is indicated by N-terminal amino acid sequencing that, such species is C-terminal fragment of collagen XVIII, with a molecular weight of about 20 kD, and such protein is named as endostatin (ES).
Studies show that the endostatin can effectively control growth, infiltration and metastases of various solid tumors, such as non-small cell lung cancer, colon cancer, brain glioma, fibroma, mesothelioma and lymphomata. Presently, endostatin is obtained through genetic engineering method. Due to too high cost of expression of endostatin by fungi, it is a trend towards utilisation of E. coli as the expression system, but the expressed protein has the problem of low renaturation and the like. To this end, the attempts to modify the structure of natural endostatin are initiated. 9 amino acids are added at the N-terminal of the natural endostatin by Luo Yongzhang, et al., and as a result, ES stability is enhanced, and half life is prolonged, bioactivity is improved, and the renaturation of the protein is higher than that of general production method. This medicament has been put into market, under the trade name of Endostar. The produced recombinant human endostatin consists of 192 amino acids, and the amino acid sequence is:
(M)GGSHHHHHHSHRDFQPVLHLVALNSPLSGGMRGIRGADFQCFQQARA VGLAGTFRAFLSSRLQDLYSIVRRADRAAVPIVNLKDELLFPSWEALFSG SEGPLKPGARIFSFDGKDVLRHPTWPQKSVWHGSDPNGRRLTESYCETWR TEAPSATGQASSLLGGRLLGQSAASCHHAYIVLCIENSFMTASK,in which, when being expressed by E. coli, Met atthe N-terminal may be deleted in some cases(SEQ ID NO. 1 or SEQ ID NO. 2).
The clinical experiments at phase III indicates that, the modified endostatin may have a certain synergetic effect when being used in combination with chemotherapeutant NP, and be capable of prolonging the tumor progression in patients.
It is confirmed by clinical experiments that the endostatin has little adverse effects and is suitable for use in combination with radiotherapy or chemotherapy. However, as the small molecule protein, the endostatin is unstable in properties, and it is indicated by studies that endostatin is likely degraded under anoxic condition (after the endostatin is co-incubated with endotheliocytes in hypoxia environment for 48 hours, the content will be decreased by about 20%). The environment inside the tumor is a hypoxia environment, thus being particularly favorable for exerting its activity. On the other hand, currently, the clinical dosage of Endostar is as high as 7.5 mg/m2, and is administrated by intravenous drip for 3-4 hours daily. Experiments show that, when being administrated by intraperitoneal injection, the endostatin is cleared in 2 hours. These indicate that, after being introduced into body, the endostatin may be hydrolyzed by the proteolytic enzyme, particularly at tumor site where the drug plays a pharmacodynamics. It is a problem to be solved urgently how to protect the protein molecule against enzymolysis and reduce clearance, thereby reducing the dosage.
Polyethylene glycol (PEG) is a safety, non-active and non-toxic polymer. By covalent attachment to the protein for chemical modification, the pegylation is able to effectively overcome the disadvantages of protein type drugs in the clinical application, for example prolong the half life of plasma, increase bioavailability, reduced protein immunogenicity, improve curative effect of drugs and safety and so on, while maintaining the biological activity of natural protein. Till now, the PEG-modified protein polypeptide drugs that have been successfully marketed include adenosine deaminase (Adagen®), asparaginase (OncasPar®), interferon α-2b (PEG-intron®), interferon α-2a (Pegasys®) etc., also more than 20 drugs, such as, uricase, hirudin, interleukin-2, interleukin-6 are under clinical studies.
During the pegylation of protein, activated PEG molecules are coupled to the free amino groups, mainly Lys, of the protein molecule. Because there is not only one free amino group in the protein chain, there are several types of modified products in the reaction products, such as, monomolecular, bimolecular, and even trimolecular products. Further, the modified products also include many types of isomers. Compared with monomolecular modified products, the loss of activity of the multimolecular modified products is higher, thus not only leading to the waste of reaction substrates, but also the reduction of overall activity of the reaction products. The problem of multimolecular modified products can be solved by controlling the reaction conditions and adopting purification means, to obtain monomolecular modified products. However, the problem of isomers in monomolecular modification still exists. Further, there may be great difference in the bioactivity of the isomers, and the mixture of the isomers is generally difficult to be purified. Therefore, it is desirable to achieve site-directed modification by controlling the reaction conditions, to promote purification and property studies of the products. Furthermore, the site-directed modification is more favorable for keeping protein activity. A method invented by Kinstler is to take advantage of lower pKa value of α-amino at the N-terminal than amino of lysine. Pegfilgrastim® is obtained by modifying granulocyte colony stimulating factor (G-CSF) with PEG acetaldehyde.